This invention relates to a method of mutually separating proteins.
There has been revealed the existence of a variety of biologically active proteins such as cytokines and peptide hormones. Recent progress of genetic engineering techniques is leading to the mass-production of such biologically active proteins and application thereof for clinical use.
These proteins, which are produced using genetic engineering techniques, often pose a problem of the necessity for separation of the end product from other substances especially when the product produced by the expression of the gene coding for the desired protein is a mixture of the desired protein and a second protein which has a methionine residue added to the amino terminus of the desired protein. The additional methionine is derived from the expression of the translational initiation codon ATG which instructs to initiate the expression of the desired gene and failure of the expression system of the host cells to remove the added methionine from the expression product. This problem is encountered both in prokaryotic and eukaryotic hosts, but more often caused in the expression of genes in prokaryotic hosts.
When both a desired protein and its N-Met derivative are produced as a mixture, it is extremely difficult to mutually separate the two proteins because the difference in physicochemical properties therebetween, if any, is very small.
The methionine residue has a molecular weight of about 131 and is moderately hydrophobic and electrically neutral because of the absence of a dissociating group. Therefore, the presence or absence of one methionine residue at the amino terminus of a macromolecule, such as a protein, having a number of dissociating groups, hydrophobic groups and hydrophilic groups, is considered to have almost no influence on the physicochemical properties of the macromolecule. Thus, it is extremely difficult to mutually separate one molecular species having a methionine residue at the amino terminus from another having no such a methionine residue.
This difficult problem is involved in a great number of proteins and becomes especially severe when Escherichia coli is used as an expression host.
There is a possibility that a protein with a methionine residue at the amino terminus differs from a protein without such a methionine residue in their higher structures, and biological activities or biological stability in vivo and in vitro. There is also a possibility that the addition of a methionine residue to the amino terminus of a protein alters the antigenicity thereof. Therefore, it is desired, from the standpoint of physiology and industrial utilization, to establish a technique for separating a protein having a methionine residue at the amino terminus from another having no such methionine residue and to isolate each protein in a substantially pure form.